Human Matrix Metalloprotease Research Articles

Covalent Labeling of Matrix Metalloproteases with Affinity-Based Probes Containing Tuned Reactive N-Acyl-N-Alkyl Sulfonamide Cleavable Linkers.

Original covalent probes with an N-acyl-N-alkyl sulfonamide cleavable linker were developed to target a broad set of human Matrix Metalloproteases (MMPs). The electrophilicity of this cleavable linker was modulated to improve the selectivity of the probes as well as reduce their unspecific reactivity in complex biological matrices. We first demonstrated that targeting the S3 subsite of MMPs enables access to broad-spectrum affinity-based probes that exclusively react with the active version of these proteases. The probes were further assessed in proteomes of varying complexity, where human MMP-13 was artificially introduced at known concentration and the resulting labeled MMP was imaged by in-gel fluorescence imaging. We showed that the less reactive probe was still able to covalently modify MMP-13 while exhibiting reduced off-target unspecific reactivity. This study clearly demonstrated the importance of finely controlling the reactivity of the NASA warhead to improve the selectivity of covalent probes in complex biological systems.

Inhibition of bacterial and human zinc-metalloproteases by bisphosphonate- and catechol-containing compounds

Compounds containg catechol or bisphosphonate were tested as inhibitors of the zinc metalloproteases, thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) which are bacterial virulence factors, and the human matrix metalloproteases MMP-9 and −14. Inhibition of virulence is a putative strategy in the development of antibacterial drugs, but the inhibitors should not interfere with human enzymes. Docking indicated that the inhibitors bound MMP-9 and MMP-14 with the phenyl, biphenyl, chlorophenyl, nitrophenyl or methoxyphenyl ringsystem in the S1′-subpocket, while these ringsystems entered the S2′- or S1 -subpockets or a region involving amino acids in the S1′- and S2′-subpockets of the bacterial enzymes. An arginine conserved among the bacterial enzymes seemed to hinder entrance deeply into the S1′-subpocket. Only the bisphosphonate containing compound RC2 bound stronger to PLN and TLN than to MMP-9 and MMP-14. Docking indicated that the reason was that the conserved arginine (R203 in TLN and R198 in PLN) interacts with phosphate groups of RC2.

Tackling Pseudomonas aeruginosa Virulence by a Hydroxamic Acid-Based LasB Inhibitor.

In search of novel antibiotics to combat the challenging spread of resistant pathogens, bacterial proteases represent promising targets for pathoblocker development. A common motif for protease inhibitors is the hydroxamic acid function, yet this group has often been related to unspecific inhibition of various metalloproteases. In this work, the inhibition of LasB, a harmful zinc metalloprotease secreted by Pseudomonas aeruginosa, through a hydroxamate derivative is described. The present inhibitor was developed based on a recently reported, highly selective thiol scaffold. Using X-ray crystallography, the lack of inhibition of a range of human matrix metalloproteases could be attributed to a distinct binding mode sparing the S1' pocket. The inhibitor was shown to restore the effect of the antimicrobial peptide LL-37, decrease the formation of P. aeruginosa biofilm and, for the first time for a LasB inhibitor, reduce the release of extracellular DNA. Hence, it is capable of disrupting several important bacterial resistance mechanisms. These results highlight the potential of protease inhibitors to fight bacterial infections and point out the possibility to achieve selective inhibition even with a strong zinc anchor.

The selectivity of galardin and an azasugar-based hydroxamate compound for human matrix metalloproteases and bacterial metalloproteases.

Inhibitors targeting bacterial enzymes should not interfere with enzymes of the host, and knowledge about structural determinants for selectivity is important for designing inhibitors with a therapeutic potential. We have determined the binding strengths of two hydroxamate compounds, galardin and compound 1b for the bacterial zinc metalloproteases, thermolysin, pseudolysin and auerolysin, known to be bacterial virulence factors, and the two human zinc metalloproteases MMP-9 and MMP-14. The active sites of the bacterial and human enzymes have huge similarities. In addition, we also studied the enzyme-inhibitor interactions by molecular modelling. The obtained Ki values of galardin for MMP-9 and MMP-14 and compound 1b for MMP-9 are approximately ten times lower than previously reported. Compound 1b binds stronger than galardin to both MMP-9 and MMP-14, and docking studies indicated that the diphenyl ether moiety of compound 1b obtains more favourable interactions within the S´1-subpocket than the 4-methylpentanoyl moiety of galardin. Both compounds bind stronger to MMP-9 than to MMP-14, which appears to be due to a larger S´1-subpocket in the former enzyme. Galardin, but not 1b, inhibits the bacterial enzymes, but the galardin Ki values were much larger than for the MMPs. The docking indicates that the S´1-subpockets of the bacterial proteases are too small to accommodate the diphenyl ether moiety of 1b, while the 4-methylpentanoyl moiety of galardin enters the pocket. The present study indicates that the size and shape of the ligand structural moiety entering the S´1-subpocket is an important determinant for selectivity between the studied MMPs and bacterial MPs.

14P MMP-2 1306 C/T promoter polymorphism and smoking: A possible role as risk factors in lung cancer

Background: Lung cancer is the leading cause of cancer death in both gender accounting for 15% of deaths among young people (0–49 years), 30% among adults (50–69 years) and 27% among those over seventy. Matrix metalloproteases (MMPs) are a large family of proteins consisting of at least 26 human MMPs. They are zincdependent endopeptidases that cleave components of the extracellular matrix (ECM) and basement membrane and has been reported that play an important role in several steps of cancer development. Many studies have shown that a nucleotide polymorphism (–1306C3T) in the MMP2 promoter in addition to smoking and age are major risk factors for the onset of lung cancer. The aim of this study was to examine the association of MMP-2 polymorphism and smoke as a risk for cancer in 71 Italians lung cancer patients. Methods: Seventy-one patients (56 men and 15 women) aged between 44 and 84 years with adeno or squamous carcinoma in inoperable stage IIIA/IIIB/IV were enrolled in this study; information about clinical and histopathological data and smoking habits were collected through the clinical charts; genotyping was performed using direct DNA sequencing. Results: 44/71 patients (62%) had the CC genotype, 15/71 (21%) patients had CT genotype and 12/71 (16%) had TT genotype. About smoking 47/71 (66%) patients were smokers, 5/71 (7%) patients had never smoked and 19/71 (26%) of them were ex-smokers and had stopped for at least 3 years. In subgroup of smokers patients, the overall average number of years of smoke was 44, the average number of smoked cigarettes was 25 and the number of pack-year 48; the smoker group was divided into heavy smokers smoking from 50 to 60 cigarettes/day, a group of heavy smokers smoking from 11 to 49 cigarettes/day and light smokers smoking from 2 to 10 cigarettes/day. A higher allelic frequency of CC genotype and stage IV disease in the heavy and mean smoking group were observed compared to light smokers (p < 0.001 and p < 0.0001 respectively) while the TT genotype was much more frequent in stage III and in subgroup of light smokers (p < 0.05). Conclusions: These preliminary results suggest a significant association between the mmp2-1306c/t polymorphism and smoke that could represent a significant risk factors of developing lung cancer. Legal entity responsible for the study: IRCCS Istituto Tumori Giovanni Paolo II Funding: Has not received any funding Disclosure: All authors have declared no conflicts of interest.

Hyaluronic acid alkyl derivative: A novel inhibitor of metalloproteases and hyaluronidases

Extracellular matrix (ECM) degradation, one of the main features of osteoarthritis, is driven by at least two major classes of enzymes: matrix metalloproteases (MMPs) and hyaluronidases. Among certain glycosaminoglycans, including natural and chemically cross-linked HAs, which are currently used as viscosupplements, the hyaluronic acid (HA) alkyl-amides (Hyadd) were here selected as the strongest MMP and hyaluronidase inhibitors. We used C. histolyticum collagenase (ChC) and bovine testicular hyaluronidase (BTH) as representative models of human MMPs and hyaluronidases, respectively. The role of the alkyl moiety was investigated using HA derivatives with varying alkyl lengths and degrees of derivatization. The selected compound was then screened against 10 different human MMPs in vitro, and the results were validated ex vivo in human synovial fluid. Hyadd-C16, identified as a lead compound, showed the highest inhibition potency against MMP13 and MMP8. The in vitro results were confirmed by the inhibition of human MMP13 (Ki=106.1μM) and hyaluronidase-2 in the synovial fluid of patients with osteoarthritis. This study demonstrates the unique properties of Hyadd-C16, including its remarkable enzymatic inhibitory activity, which is conferred by the hydrophobic chain, and its high biocompatibility and water solubility of the HA backbone.

Computational and biochemical design of a nanopore cleavable by a cancer-secreted enzyme.

Many proteinaceous macromolecules selectively transport substrates across lipid bilayers and effectively serve as gated nanopores. Here, we engineered cleavage-site motifs for human matrix metalloprotease 7 (MMP-7) into the extracellular and pore-constricting loops of OprD, a bacterial substrate-specific transmembrane channel. Concurrent removal of two extracellular loops allowed MMP-7 to access and hydrolyze a cleavage-site motif engineered within the pore's major constricting loop, in both membrane-incorporated and detergent-solubilized OprDs. Import of antibiotics by the engineered OprDs into living bacteria pointed to their proper folding and integration in biological membranes. Purified engineered OprDs were also found to be properly folded in detergent. Hence, this study demonstrates the design of nanopores with a constriction cleavable by tumor-secreted enzymes (like MMP-7) for their potential incorporation in lipid-based nanoparticles to accelerate drug release at the tumor site.

Family-wide characterization of matrix metalloproteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity

MMPs (matrix metalloproteases) are a family of zinc-dependent endopeptidases widely distributed throughout all kingdoms of life. In mammals, MMPs play key roles in many physiological and pathological processes, including remodelling of the extracellular matrix. In the genome of the annual plant Arabidopsis thaliana, five MMP-like proteins (At-MMPs) are encoded, but their function is unknown. Previous work on these enzymes was limited to gene expression analysis, and so far proteolytic activity has been shown only for At1-MMP. We expressed and purified the catalytic domains of all five At-MMPs as His-tagged proteins in Escherichia coli cells to delineate the biochemical differences and similarities among the Arabidopsis MMP family members. We demonstrate that all five recombinant At-MMPs are active proteases with distinct preferences for different protease substrates. Furthermore, we performed a family-wide characterization of their biochemical properties and highlight similarities and differences in their cleavage site specificities as well as pH- and temperature-dependent activities. Detailed analysis of their sequence specificity using PICS (proteomic identification of protease cleavage sites) revealed profiles similar to human MMPs with the exception of At5-MMP; homology models of the At-MMP catalytic domains supported these results. Our results suggest that each At-MMP may be involved in different proteolytic processes during plant growth and development.

99mTc-MAG3-Aptamer for Imaging Human Tumors Associated with High Level of Matrix Metalloprotease-9

The human matrix metalloprotease 9 (hMMP-9) is involved in many physiological processes such as tissue remodeling. Its overexpression in tumors promotes the release of cancer cells thus contributing to tumor metastasis. It is a relevant marker of malignant tumors. We selected an RNA aptamer containing 2'-fluoro, pyrimidine ribonucleosides, that exhibits a strong affinity for hMMP-9 (K(d) = 20 nM) and that discriminates other human MMPs: no binding was detected to either hMMP-2 or -7. Investigating the binding properties of different MMP-9 aptamer variants by surface plasmon resonance allowed the determination of recognition elements. As a result, a truncated aptamer, 36 nucleotides long, was made fully resistant to nuclease following the substitution of every purine ribonucleoside residue by 2'-O-methyl analogues and was conjugated to S-acetylmercaptoacetyltriglycine for imaging purposes. The resulting modified aptamer retained the binding properties of the originally selected sequence. Following (99m)Tc labeling, this aptamer was used for ex vivo imaging slices of human brain tumors. We were able to specifically detect the presence of hMMP-9 in such tissues.

Extra- and Intracellular Imaging of Human Matrix Metalloprotease 11 (hMMP-11) with a Cell-penetrating FRET Substrate

Matrix metalloprotease 11 (MMP-11), a protease associated with invasion and aggressiveness of cancerous tissue, was postulated as a prognostic marker for pancreatic, breast, and colon cancer patients. Expression analysis, however, did not reveal localization and regulation of this protease. Thus, cellular tools for the visualization of MMP-11 are highly desirable to monitor presence and activity and to elucidate the functional role of MMP-11. Therefore, fluorescein-Dabcyl-labeled Foerster resonance energy transfer (FRET) substrates were developed. The design focused on enhanced peptide binding to human MMP-11, employing an unusual amino acid for the specificity pocket P1'. The addition of several arginines resulted in a cell-permeable FRET substrate SM-P124 (Ac-GRRRK(Dabcyl)-GGAANC(MeOBn)RMGG-fluorescein). In vitro evaluation of SM-P124 with human MMP-11 showed a 25-fold increase of affinity (k(cat)/K(m) = 9.16 × 10(3) m(-1) s(-1), K(m) = 8 μm) compared with previously published substrates. Incubation of pancreatic adenocarcinoma cell line MIA PaCa-2 and mamma adenocarcinoma cell line MCF-7 with the substrate SM-P124 (5 μm) indicated intra- and extracellular MMP-11 activity. A negative control cell line (Jurkat) showed no fluorescent signal either intra- or extracellularly. Negative control FRET substrate SM-P123 produced only insignificant extracellular fluorescence without any intracellular fluorescence. SM-P124 therefore enabled intra- and extracellular tracking of MMP-11-overexpressing cancers such as pancreatic and breast adenocarcinoma and might contribute to the understanding of the activation pathways leading to MMP-11-mediated invasive processes.

Ancylostoma ceylanicum Excretory–Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory–secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 Å resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2′s netrin-like fold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

Physiology and pathophysiology of matrix metalloproteases

Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn2+ ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with respect to substrate specificity, cellular and tissue localization, membrane binding and regulation that make this a very versatile family of enzymes with a multitude of physiological functions, many of which are still not fully understood. Essentially, all members of the MMP family have been linked to disease development, notably to cancer metastasis, chronic inflammation and the ensuing tissue damage as well as to neurological disorders. This has stimulated a flurry of studies into MMP inhibitors as therapeutic agents, as well as into measuring MMP levels as diagnostic or prognostic markers. As with most protein families, deciphering the function(s) of MMPs is difficult, as they can modify many proteins. Which of these reactions are physiologically or pathophysiologically relevant is often not clear, although studies on knockout animals, human genetic and epigenetic, as well as biochemical studies using natural or synthetic inhibitors have provided insight to a great extent. In this review, we will give an overview of 23 members of the human MMP family and describe functions, linkages to disease and structural and mechanistic features. MMPs can be grouped into soluble (including matrilysins) and membrane-anchored species. We adhere to the ‘MMP nomenclature’ and provide the reader with reference to the many, often diverse, names for this enzyme family in the introduction.

Combinatorial gene therapy renders increased survival in cirrhotic rats

BackgroundLiver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by acummulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals.MethodsWistar rats (80 g) underwent chronic intoxication with CCl4: mineral oil for 8 weeks. Cirrhotic animals were injected with a combined dose of Ad-delta-huPA plus Ad-MMP8 (3 × 1011 and 1.5 × 1011 vp/Kg, respectively) or with Ad-beta-Gal (4.5 × 1011) and were killed after 2, 4, 6, 8 and 10 days. Then, liver and serum were collected. An additional set of cirrhotic animals injected with combined gene therapy was also monitored for their probability of survival.ResultsOnly the cirrhotic animals treated with therapeutical genes (Ad-delta-huPA+Ad-MMP-8) showed improvement in liver fibrosis. These results correlated with hydroxyproline determinations. A significant decrement in alpha-SMA and TGF-beta1 gene expression was also observed. Cirrhotic rats treated with Ad-delta-huPA plus Ad-MMP8 had a higher probability of survival at 60 days with respect to Ad-beta-Gal-injected animals.ConclusionA single administration of Ad-delta-huPA plus Ad-MMP-8 is efficient to induce fibrosis regression and increase survival in experimental liver fibrosis.

Antimicrobial Activity of a Halocidin-Derived Peptide Resistant to Attacks by Proteases

Cationic antimicrobial peptides (AMPs) have attracted a great deal of interest as a promising candidate for a novel class of antibiotics that might effectively treat recalcitrant infections caused by a variety of microbes that are resistant to currently available drugs. However, the AMPs are inherently limited in that they are inevitably susceptible to attacks by proteases generated by human and pathogenic microbes; this vulnerability severely hinders their pharmaceutical use in human therapeutic protocols. In this study, we report that a halocidin-derived AMP, designated HG1, was found to be resistant to proteolytic degradation. As a result of its unique structural features, HG1 proved capable of preserving its antimicrobial activity after incubation with trypsin, chymotrypsin, and human matrix metalloprotease 7 (MMP-7). Additionally, HG1 was observed to exhibit profound antimicrobial activity in the presence of fluid from human skin wounds or proteins extracted from the culture supernatants of Staphylococcus aureus and Pseudomonas aeruginosa. Greater understanding of the structural motifs of HG1 required for its protease resistance might provide feasible ways to solve the problems intrinsic to the development of an AMP-based antibiotic.

Neuroprotective and MMP-9 inhibitory activity of hydroethanolic extract of Arbustus unedo leaves

Arbutus unedo L., the strawberry tree (Ericaceae family), is an endemic Mediterranean species. Its leaves have been employed for a long time in traditional and popular medicine as an astringent, diuretic, urinary anti-septic, and more recently, in the therapy of hypertension and diabetes [1]. The aim of this work is to evaluate the antioxidant properties of a hydroethanolic extract of A. unedo leaves in a neurodegeneration cell model and the inhibitory activity to human matrix metalloprotease (MMP-9), an enzyme involved in cancer invasion.

Identification in human osteoarthritic chondrocytes of proteins binding to the novel regulatory site AGRE in the human matrix metalloprotease 13 proximal promoter

Matrix metalloprotease 13 (MMP-13) plays a major role in osteoarthritic (OA) processes. We previously identified the AG-rich element (AGRE) regulatory site (GAAAAGAAAAAG) in the proximal promoter of this gene. Electrophoretic mobility shift assays (EMSAs) done with nuclear extracts from OA chondrocytes showed the presence of 2 AGRE protein-binding complexes, the formation of which depended on the pathophysiologic state (high or low) of the cells; the low OA (L-OA) chondrocytes have low MMP-13 basal levels and high interleukin-1beta (IL-1beta) inducibility, and the high OA (H-OA) chondrocytes have high MMP-13 basal levels and low IL-1beta inducibility. In this study, we sought to determine the importance of individual AGRE bases in promoter activity and to identify AGRE binding proteins from L-OA and H-OA chondrocyte complexes. Promoter activity was determined following transient transfection into human OA chondrocytes. AGRE binding proteins were identified by mass spectroscopy. Individual mutations of the AGRE site differentially modulated promoter activity, indicating that the intact AGRE site is required for optimal MMP-13 expression. Damage-specific DNA binding protein 1 (DDB-1) was identified in the L-OA chondrocyte-binding complex. EMSA experiments performed with the mutation of the left AGRE site (GTGCTGAAAAAG) and nuclear extracts of L-OA chondrocytes reproduced the pattern seen in the H-OA chondrocytes. Mass spectroscopy identified p130cas as one of the proteins in this complex. Supershift experiments showed the presence of p130cas and nuclear matrix transcription factor 4 (NMP-4) in the wild-type AGRE/H-OA chondrocyte complex. These data suggest that the binding of p130(cas) and NMP-4 to the AGRE site regulates MMP-13 expression and may trigger the change in human chondrocytes from the L-OA state to the H-OA state.

Platelet adherence to isolated rat hepatic sinusoidal endothelial cells after cold preservation.

Platelet adhesion to hepatic sinusoidal endothelial cells (SEC) is a major mechanism of cold preservation injury. This study was performed to determine whether cold preservation leads to adhesion between SEC and platelets in the absence of other cell types, whether adherent platelets become activated upon adhesion, and whether there is increased expression of the platelet receptor von Willebrand factor (vWF) on cold preserved SEC. Because we previously showed that cold causes actin disassembly and matrix metalloprotease (MMP) secretion by SEC, we also questioned whether these events are related to increased adhesion of platelets to SEC after preservation. Isolated SEC were cold preserved for 8 hr and rewarmed briefly. Biotinylated platelets were added to the plates and adhesion was assessed. Activation of platelets was determined by staining with antibody to P-selectin. Expression of vWF was assessed with a specific antibody. Cold preservation induced increased adhesion of platelets to SEC in the absence of other cell types. Adherent platelets were activated. Preservation increased the expression of vWF on SEC. Pretreatment with phalloidin or treatment with MMP inhibitors partly prevented platelet adhesion and activation, as well as vWF expression. Treatment of SEC at 37 degrees C with recombinant human MMPs for 24 hr also rendered the SEC more adherent for unactivated platelets. Cold preservation of SEC results in increased expression of the platelet receptor vWF, increased platelet adhesion, and platelet activation. This is at least partly mediated by actin disassembly and MMP secretion.

Human matrix metalloprotease activation by insults of bacterial infection involving proteases and free radicals.

We found that human matrix metalloproteases (MMPs) may be processed from their proenzyme forms (proMMP) to their active forms by two new and unique mechanisms: Firstly, by bacterial proteases such as Pseudomonas elastase and Vibrio cholerae protease, which cleave off the N-terminal autoinhibitory domain (so-called cysteine switch) from proMMPs. The second mechanism depends on free radical generation by activated polymorphonuclear leukocytes (PMNs). In this case, peroxynitrite (ONOO-) or nitrogen dioxide radical (.NO2), the reaction products of either superoxide (O2.-) or molecular oxygen (O2) and nitric oxide (.NO), are the key reactants. Both O2.- and .NO are generated by activated macrophages and PMNs as a result of immunologic responses involving various proinflammatory cytokines. .NO2 or ONOO- seems to interact with a single cysteine residue in the propeptide autoinhibitory domain, or so-called cysteine switch of proMMPs, thus transforming proMMPs into their active conformation. Furthermore, reactive oxygen species are known to inactivate the alpha1-protease inhibitor (alpha1-PI), a potent neutrophil elastase inhibitor in plasma. In addition, we found that such radicals activate MMPs which degrade and inactivate alpha1-PI by proteolysis. Thus, the activation of MMPs, accompanied by the inactivation of alpha1-PI, will bring about enhanced proteolytic damage to the matrix tissues of the infected sites by both MMPs and elastase.

Asymmetric synthesis of a conformationally constrained N-phosphonoalkyl dipeptide

An asymmetric synthesis has been developed to prepare a phosphono-protected (2 S ,6 R )-6-phosphono-piperidine-2-carboxylic acid and its (2 R , 6 S ) isomer. Coupling qith tryptophan derivative afforded conformationally constrained N -phosphonoalkyl dipeptides ( S,S,R )- 2 and ( S,R,S )- 14 which have been both evaluated as human matrix metalloprotease inhibitors. An asymmetric synthesis has been developed to prepare phosphono-protected (2 S ,6 R )-6-phosphono-piperidine-2-carboxylic acid and its (2 R , 6 S ) isomer. Coupling with tryptophan derivative afforded conformationally constrained N -phosphonoalkyl dipeptides ( S,S,R )- 2 and ( S,R,S )- 14 which have been both evaluated as human matrix metalloprotease inhibitors